The proposed work will explore the genetic basis for real-world variability in the behavior of human pathogens during infections of individual humans and whole populations. The focus will be on two gramnegative pathogens, Burkholderia pseudomallei and Shigella dysenteriae, both of which cause infectious diseases associated with substantial mortality in humans. B. pseudomallei is an opportunistic pathogen that causes melioidosis, a disease largely found in Southeast Asia and Northern Australia; it has been classified as a select agent because of its perceived potential as a biowarfare agent. S. dysenteriae causes dysentery epidemics, predominantly in developing countries, which cause substantial mortality, particular in children. The basic hypothesis behind the proposed work is that genetic changes occurring during individual B. pseudomallei infections or during S. dysenteriae epidemics will reveal the selective pressures operating on these species as a result of host defenses, treatment, andin the case of S. dysenteriaethe dynamic changes in the characteristics of the host population within which the epidemic is spreading. A better understanding of the molecular basis for these genetic effects may suggest new approaches to the clinical and public-health management of the diseases. The project has a major technical component since, although new-generation-sequencing technology is opening up the opportunity for studies of the type proposed, key aspects of the technology remain underdeveloped. We place particular emphasis on improvements in the rapidity with which new base-pair-accurate, finished genome sequences can be produced for the index strains of particular infections or epidemics. These reference sequences play a key in role in the experimental and computational methods used to detect the genetic changes that occur during infections and epidemics.